The objective of this proposal is to elucidate the mechanisms whereby two xenobiotics activate programmed cell death or apoptosis (APOP). We have developed two cell culture models of drug-induced APOP, namely, 1) rat hepatocytes treated with microtubule antagonists vinblastine (VBL) or colchicine (COL), and 2) L929 mouse fibroblasts treated with the topoisomerase II inhibitor etoposide. In both models, cell killing is accompanied by DNA fragmentation and is prevented by the inhibition of either protein synthesis or protein kinase C (PKC). Furthermore, the killing of hepatocytes by VBL or COL requires extracellular calcium, is preceded by transcription of the gene for p53, and is accompanied by evidence of transglutaminase (tTG) activity. We propose that drug-induced APOP represents a programmed reaction to functional inactivation of the cell. In turn, the cell recognizes the particular injury and responds with biochemical events characteristic of APOP, including DNA fragmentation and tTG activity. We propose that the cross-linking of membrane components by the tTG compromises the barrier function of the plasma membrane, an event that heralds the death of the cell. By contrast, DNA fragmentation is a marker, rather than the sine qua non, of APOP. To analyze this hypothesis, we will pursue the following aims: 1) characterization of the time course of the induction of tTG activity and the determination of its relationship to the loss of viability; 2) characterization of the changes in the pattern of protein synthesis; 3) determination of how, when, and where alterations of Ca2+ homeostasis promote drug-induced APOP; 4) similar determination of how, when and where the activation of PKC is related to the pathogenesis of cell death in our models of APOP; and 5) determination of the significance with respect to cell death of the activation of p53 gene transcription.